Optimization of human serum albumin production in methylotrophic yeast Pichia pastoris by repeated fed-batch fermentation

An optimization method for repeated fed-batch fermentation was established with the aim of improving the recombinant human serum albumin (rHSA) production in Pichia pastoris. A simulation model for fed-batch fermentation was formulated and the optimal methanol-feeding policy calculated by dynamic programming method using five different methanol-feeding periods. The necessary state variables were collected from the calculated results and used for further optimization of repeated fed-batch fermentation. The optimal operation policy was investigated using the pre-collected state variables by estimating the overall profit per total methanol-feeding time. The calculated results indicated that the initial cell mass from the 2nd fed-batch fermentation on should be set at 35 or 40 g and methanol-feeding time at 264 h. In repeated fed-batch fermentation using the optimal operation policy, actual culture volume was in good agreement with the values simulated by model equations, but some discrepancy was observed in rHSA production. Minimum experiments were therefore carried out to re-evaluate rHSA production levels, which were then applied in re-calculations to determine the optimal operation policy. The optimal policy for repeated fed-batch fermentation established in the present study (i.e., 4-times-repeated fed-batch fermentation) achieved a 47% increase in annual rHSA production. Optimization of the culture period also brought about a 28% increase in annual rHSA production even in simple (not repeated) fed-batch fermentation.     

Formation of ceramide-enriched domains in lipid particles enhances the binding of apolipoprotein E

We investigated the interaction between apolipoprotein E (apoE) and ceramide (CER)-enriched domains on the particles, by using lipid emulsions containing sphingomyelin (SM) or CER as model particles of lipoproteins. The sphingomyelinase (SMase)-induced aggregation of emulsion particles was prevented by apoE. CER increased the amount of apoE bound to emulsion particles. The confocal images of CER-containing large emulsions with two fluorescent probes showed three-dimensional microdomains enriched in CER. SMase also induced the formation of CER-enriched domains. We propose apoE prefers to bind on CER-enriched domains exposed on particle surface, and thus inhibits the aggregation or fusion of the particles.     

Use of 13C chemical shift surfaces in the study of carbohydrate conformation. Application to cyclomaltooligosaccharides (cyclodextrins) in the solid state and in solution

The anomeric carbon chemical shifts of free cyclomaltohexaose, -heptaose, -octaose, -decaose, and -tetradecaose (alpha-, beta-, gamma-, epsilon-, and eta-cyclodextrin, respectively), and of alpha-cyclodextrin inclusion complexes, both in the solid state and in solution, were computed using ab initio 13C chemical shift surfaces for the D-Glcp-alpha-(1-->4)-D-Glcp linkage as a function of the glycosidic bond dihedral angles. Chemical shift calculations in the solid state used angle pairs measured from cyclodextrin X-ray structures as input. For estimations in the liquid state two different approaches were employed to account for dynamic averaging. In one, the computed solid-state anomeric carbon chemical shifts for each cyclodextrin D-Glcp monomer were simply averaged to obtain an estimate of the 13C shifts in solution. In the other, chemical shifts for the anomeric carbons were determined by averaging back-calculated 13C shift trajectories derived from a series of 5 ns molecular dynamic simulations for the oligosaccharides with explicit representation of water. Good agreement between calculated and experimental 13C shifts was found in all cases. Furthermore, our results show that the ab initio 13C chemical shift surfaces are sufficiently sensitive to reproduce the small variations observed for the anomeric 13C shifts of the different cyclodextrin D-Glcp units in the solid state with excellent accuracy. The use of chemical shift surfaces as tools in conformational studies of oligosaccharides is discussed.     

超声彩色血流成像的计算机快速仿真方法

研究超声彩色血流成像的快速仿真方法,克服原先仿真方法非常耗时的缺点。方法超声彩色血流成像计算机仿真中,血流信号是对成像区间内所有点散射体的回波信号累加而得到的。通过引入新的等效散射体模型,可以大大降低散射体的密度,从而减少计算回波信号所需时间。在计算机上用Matlab编程来进行仿真实验,对以往仿真方法和基于等效散射体模型方法的性能进行比较。结果实验表明:基于等效散射体模型的仿真,在保证相同流速精度的前提下,仿真速度比传统方法提高了10倍以上。结论基于等效散射体模型的仿真方法能极大地提高超声彩色血流成像的仿真速度,可以为超声彩色血流成像的方法研究提供便利。     

POWSIM: a computer program for assessing statistical power when testing for genetic differentiation

Knowledge of statistical power is essential for sampling design and data evaluation when testing for genetic differentiation. Yet, such information is typically missing in studies of conservation and evolutionary genetics, most likely because of complex interactions between the many factors that affect power. powsim is a 32‐bit Windows/DOS simulation‐based computer program that estimates power (and α error) for chi‐square and Fisher's exact tests when evaluating the hypothesis of genetic homogeneity. Optional combinations include the number of samples, sample sizes, number of loci and alleles, allele frequencies, and degree of differentiation (quantified as F_ST). powsim is available at http://www.zoologi.su.se/~ryman .     

Dynamic energy budget theory restores coherence in biology

We present the state of the art of the development of dynamic energy budget theory, and its expected developments in the near future within the molecular, physiological and ecological domains. The degree of formalization in the set-up of the theory, with its roots in chemistry, physics, thermodynamics, evolution and the consistent application of Occam's razor, is discussed. We place the various contributions in the theme issue within this theoretical setting, and sketch the scope of actual and potential applications.     

Structure prediction and molecular simulation of gases diffusion pathways in hydrogenase

Although hydrogen is considered to be one of the most promising future energy sources and the technical aspects involved in using it have advanced considerably, the future supply of hydrogen from renewable sources is still unsolved. The [Fe]- hydrogenase enzymes are highly efficient H(2) catalysts found in ecologically and phylogenetically diverse microorganisms, including the photosynthetic green alga, Chlamydomonas reinhardtii. While these enzymes can occur in several forms, H(2) catalysis takes place at a unique [FeS] prosthetic group or H-cluster, located at the active site. 3D structure of the protein hydA1 hydrogenase from Chlamydomonas reinhardtti was predicted using the MODELER 8v2 software. Conserved region was depicted from the NCBI CDD Search. Template selection was done on the basis NCBI BLAST results. For single template 1FEH was used and for multiple templates 1FEH and 1HFE were used. The result of the Homology modeling was verified by uploading the file to SAVS server. On the basis of the SAVS result 3D structure predicted using single template was chosen for performing molecular simulation. For performing molecular simulation three strategies were used. First the molecular simulation of the protein was performed in solvated box containing bulk water. Then 100 H(2) molecules were randomly inserted in the solvated box and two simulations of 50 and 100 ps were performed. Similarly 100 O(2) molecules were randomly placed in the solvated box and again 50 and 100 ps simulation were performed. Energy minimization was performed before each simulation was performed. Conformations were saved after each simulation. Analysis of the gas diffusion was done on the basis of RMSD, Radius of Gyration and no. of gas molecule/ps plot.     

A stochastic model of neuronal growth cone guidance regulated by multiple sensors

Neuronal growth cones migrate directionally under the control of axon guidance molecules, thereby forming synapses in the developing brain. The signal transduction system by which a growth cone detects surrounding guidance molecules, analyzes the detected signals, and then determines the overall behavior remains undetermined. In this study, we describe a novel stochastic model of this behavior that utilizes multiple sensors on filopodia to respond to guidance molecules. Overall growth cone behavior is determined by using only the concentration gradients of guidance molecules in the immediate vicinity of each sensor. The detected signal at each sensor, which is treated as a vector quantity, is sent to the growth cone center and then integrated to determine axonal growth in the next step by means of a simple vector operation. We compared the results of computer simulations of axonal growth with observations of actual axonal growth from co-culture experiments using olfactory bulb and septum. The probabilistic distributions of axonal growth generated by the computer simulation were consistent with those obtained from the culture experiments, indicating that our model accurately simulates growth cone behavior. We believe that this model will be useful for elucidating the as yet unknown mechanisms responsible for axonal growth in vivo.     

Activation of the Ghrelin Receptor is Described by a Privileged Collective Motion: A Model for Constitutive and Agonist-induced Activation of a Sub-class A G-Protein Coupled Receptor (GPCR)

Three homology models of the human ghrelin receptor (GHS-R1a) have been generated from the available X-ray structures of rhodopsin (RHO model), opsin (OPS model) and beta-2 adrenergic receptor (B2 model). The latter was used as a starting point for combined molecular dynamics simulation (MDS) and full atom normal modes analysis (NMA). A low-frequency normal mode (mode 16) perfectly reproduced the intracellular motions observed between B2 and RHO models; in the opposite direction along the same mode, the generated structures are closer to the OPS model, suggesting a direct link with GHS-R1a activation. This was in agreement with motions of the seven transmembranous segments, increase of the solvent accessibility of the 140-ERY-142 sequence, and flip of the Trp276 (C WLP) residue, some features related to GPCRs activation. According to our model, His280 was proposed to stabilize Trp276 in the active state; this was verified by site-directed mutagenesis and biochemical characterization of the resulting H280A and H280S mutants, which were fully functional but sharing an important decrease of their basal activities. Docking performed with short ghrelin derivatives Gly-Ser-Ser _[octa]-Phe-NH ₂ and Gly-Ser-Ser _[octa]-Phe-Leu-NH ₂ allowed the identification of a robust position of these peptides in the active site of the receptor. This model was refined by MDS and validated by docking experiments performed on a set of 55 ghrelin receptor ligands based on the 1,2,4- triazole scaffold. Finally, NMA performed on the obtained peptide-receptor complex suggested stabilization of the Trp276 residue and of the whole receptor in the active state, preventing the motion observed along mode 16 computed for the unbound receptor. Our results show that NMA offers a powerful approach to study the conformational diversity and the activation mechanism of GPCRs.